Exam_3_review_questions_answersA

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Review Sheet for Bio 230, Exam 3 1) What is the general function of transcription factors in cells, and how do they accomplish this function? Transcription factors are small proteins that bind to specific DNA sequences to facilitate or suppress transcription. They can accomplish this by interacting with other transcription machinery. 2) Describe one example of negative control of gene expression. The repressor in the lac operon or the tryptophan in the repressible operon exert negative control because they repress transcription. There are many other potential examples. 3) In a repressible operon such as the regulation of tryptophan metabolism, how would you cause the repressor to inactivate? Decrease the [corepressor]. 4) Every cell with a nucleus within a multicellular organism contains the same DNA within its nucleus. Describe how the major differences between cell types originate, meaning, how does the same genome lead to many different cell types? The differentiation of eukaryotic cells is due to the differential expression of genes and their translated proteins at different times and in different combinations. 5) Describe a technique that you would use to measure the mRNA that is expressed in a cell. One possibility is DNA microarrays. For these, mRNA extracted from cells are converted to cDNA by reverse transcriptase. The cDNA is then mixed with fluorescent probes that attach to one end of the cDNA. Often there is an amplification step to increase the number of copies (hopefully maintaining same proportions of copies) using the polymerase chain reaction (PCR). The cDNA mixture is then added to well of the DNA microarray (gene chip). Each well of the microarray contains a complementary DNA sequence for one gene. If that gene is present in the sample, it should bind in the well. Then unbound cDNA is washed away. The DNA microarray is imaged, and fluorescently labeled wells indicate that the gene in the well was present in the cDNA sample. 6) Name one modification of histones and describe the histone code. Histone tails can be methylated, acetylated or phosphorylated on their tails on specific amino acids. The histone code describes the series of modifications on histone tails that contribute to controlling whether DNA can be transcribed. 7) What is DNA wrapped around histones called and what is its function? a. It is called a nucleolus and it helps to initiate translation. b. It is called a nucleolus and it helps to compact DNA. c. It is called a nucleosome and it helps to initiate translation. d. It is called a nucleosome and it helps to compact DNA.

8) Which amino acids are coded for by the codons CAC AAG GGG, and how would they be affected by changes in the third positions of the codons? His-Arg-Gly. Changing the first codon to CAA or CAG would change to glutamine, changing the second to AAU or AAC changes it to serine, and changing the nucleotide in the third position of the third codon has no effect (always codes for glycine). 9) How will translation be affected if a cell is unable to synthesize or obtain methionine? It will not be possible to initiate translation, since translated is initiated by formylated methionine (fMet) binding in the P site of the ribosome. 10) After the initiation of translation, which site in the ribosome do charged tRNAs enter and bind? After the initial binding of methionine in the P site, all subsequent charged tRNAs bind in the A site. 11) Name a post-transcriptional modification and state what role that it serves. Addition of a 5’ methylguanosine cap or addition of a 3’ series of adenine (polyadenylation). Both stabilize the mRNA and prevent degradation. 12) Why are immature mRNAs much longer than mature mRNAs? They still contain intron (non-protein coding) segments. 13) Where does RNA polymerase bind to initiate transcription in prokaryotes or eukaryotes? The promoter sequence. 14) In this picture of a DNA microarray (gene chip), why are some spots colored red, yellow or green, and others are colored gray? Assume that there were two cDNA samples that were tagged with red (Sample 1) and green (Sample 2) fluorescent probes, respectively. Then red spots indicate that a gene was expressed in sample 1 but not 2, green spots indicate that a gene was expressed in 2 but not 1, yellow indicates that a gene was expressed in both samples, and gray spots indicate a gene that was not expressed in either sample.

15) The diagram to the right displays the time course of mRNA expression for genes in involved in the TCA cycle. Below that are two solid lines, with the dashed lines representing baseline gene expression, and upwards from that line is considered higher gene expression, with time along the x-axis the same as the top graph. Reading the graph to the right, which genes could give the gene expression profiles shown for the top solid line or the bottom solid line? You may use the 4 letter gene name for your answer (e.g. CIT1) Only one answer is sufficient for each, even if there is more than one potential answer. For the top one, in which expression increases and then decreases, SDH3 would be one example. For the bottom one that shows a sustained increase in expression, SDH2 is probably the best choice. 16) What is the open reading frame and why is it important for gene and protein sequencing? The open reading frame (ORF) refers to the start position on the mature mRNA that dictates how the codon triplets will be read to determine the amino acid sequence. 17) What will be the consequence of inserting a uracil into the 6 th position in the following sequence. Assume that you begin in the open reading frame. (A chart will be provided for the exam). 5’ -GAG-CCC-AAA-CGC- 3’ goes to 5’ -GAG-CCU-CAA-ACG-C- 3’ giving Glu-Pro-Lys-Arg goes to Glu-Pro-Gln-Thr 18) What is a consensus sequence? This is a DNA promoter sequence whose position relative to the transcription start and whose sequence is largely conserved across species, such as the -10 sequence in bacteria or the TATA box in eukaryotes. 19) What serves as the intermediate between mRNA and protein? tRNA 20) If the 3’ end of the tRNA has been cleaved, what part of translation w ill be affected?

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The tRNA cannot be charged with an amino acid in this case. 21) How many amino acids does each codon code for? Is the number of codons for each amino acid, larger, smaller or the same? 1. The code is redundant, so multiple codons code for many of the amino acids. 22) What are the main steps in translation by the ribosome? Small subunit binds mRNA rRNA recognizes AUG on mRNA tRNA-Met enters P site and binds to AUG Large subunit binds (GTP) Second tRNA binds A site (GTP) Peptide bond (peptide attached to 2nd tRNA) Translocation by 1 codon towards 3’ end (GTP) Release of 1st tRNA from E site Continue 5-8 until stop codon is reached Stop codon in A site -> termination (GTP) 23) A space probe brings back a new life form. It is carbon-based and its genetic material is similar to Earth DNA except that it uses 6 different nucleotide bases instead of 4. Its proteins are also similar to those on Earth, but there are 28 amino acids instead of 20. You hypothesize about the genetic code for this organism. How many "letters" would you propose in each codon of the genetic code for this organism? The code should be unambiguous and may be degenerate. A minimum codon length of 2 would be appropriate, giving 6x6=36 codons, enough to be unambiguous and include start and stop codons. A longer codon would work, but it would be highly degenerate in that case, since a triplet length would give 216 codons. 24) Which steps in translation require ATP hydrolysis? Which steps require GTP hydrolysis? The only part of translation that requires ATP hydrolysis is charging the tRNA. The GTP requiring steps are listed in the answer to 22. 25) Construct a genetic AND, OR, and NAND logic circuit. For an AND, you want two components to come together to form a transcriptional activator. For an OR, you want two components that each individually are transcriptional activators and that don’t interfere with each other. For a NAND, you can have something similar to the repressible operon, using a corepressor. When either component of the corepressor is present/active by itself, transcription will not be affected. It is only when the two components come together to form an active repressor that transcription will be blocked.

26) In the inducible operon, what would be an example of positive control? Of negative control? Which components would constitute an AND circuit? cAMP or cAMP-cRP is a form of positive control. Glucose and lactase (because it breaks down the inducer lactose) are forms of negative control. cAMP and lactose together form an AND circuit for induction of transcription here. 27) In the repressible operon, what would be an example of negative control? Which components would constitute a NAND circuit? Tryptophan is an example of negative control, and as mentioned above, the corepressor can be considered a NAND circuit. 28) Describe how a DNA microarray works. Complementarity between DNA and see answer to 5. 29) Which histone modifications permit or repress transcription and where on the histone are the modifications made? Acetylation permits transcription while methylation and deacetylation represses transcription . Modifications are made on the histone tails. 30. Give a biological example of passive diffusion, facilitated diffusion, and active transport. Using examples we discussed in class, oxygen and water can passively diffuse across the membrane, glucose can pass through via transporters, and Na and K are transported against their gradients by active transport. Other well-described examples would be acceptable. 31. A membrane is permeable only to water. It is placed in a solution with 150 mM KCl inside and 15 mM KCl outside. What happens? In this case, inside the membrane is hypotonic and water will flow in to balance the concentration. 32. From the question above, what is the equilibrium potential if the membrane is permeable only to Cl-? To K+? to both? We can compute the equilibrium potential from the Nernst equation Eion=- (60/z)*log10[Cin/Cout]. Note that this is the same as Eion=(60/z)*log10[Cout/Cin]. z=-1 for Cl-, so ECl=-(60/-1)*log10(150/15)=60*log10(10)=60*1=+60 mV. In a typical cell, there is typically ~150 mM extracellular NaCl and intracellular [Cl-]~10-15 mM (KCl is not normally found at high concentrations in the cell), so ECl is about -60 to -70 mV. For K+, z=1, so EK=-(60/+1)*log10(150/15)=-60*log10(10)=-60*1=-60 mV. so EK

in this example is -60 mV. In a typical cell, EK is closer to -90 mV. If the cell is permeable to both, the combined reversal potential would depend on the relative permeabilities of potassium and chloride. If they were equal, the reversal potential would be 0. 33. In ion channels, what is the name of the region that determines ion permeability? It is called the selectivity filter. 34. In ion channels, what are two common gating mechanisms that control whether a channel is open or closed? Membrane voltage, ligand binding, pH, and mechanical deformation are all potential mechanisms. For example, Shaker K+ and action potential K+ channels are voltage-gated channels, such that depolarization of the membrane will open the channel. 35. What initially changes the channel conformation for the Na/K pump? ATP hydrolysis changes the channel conformation. 36. Why do K+ ions dissociate from the Na/K pump? After dephosphorylation, the channel returns to the E1 conformation, lowering the affinity of the pump for K+, so they dissociate. 37. Give one biological example of a symport and one example of an antiport. The Na/glucose transport is an example of a symport and the Na/K exchange or the H/K exchange is an example of an antiport 38. Name two factors that determine transition temperature. Number of unsaturated bonds and fatty acid chain length? 39. How might you test that a protein is associated with a lipid raft ? Lipid raft membranes have a different composition than standard membranes. If those membranes can be isolated or identified, an antibody to the protein would determine whether the protein is colocalized specifically in the lipid raft regions. 40. If you wanted to measure membrane fluidity, what is one way that you could do it? FRAP 41. How would you determine whether a portion of a protein is extracellular or cytoplasmic? Bathe some cells in trypsin and some not. Isolate protein and run a gel. Determine which bands on gel have shifted .

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